The objective of this application is to develop the career of Dr. Christian Badr to facilitate the transition to a stable independent phase and attain his ultimate goal to become an independent scientist and establish his own research program. The career development plan created by the candidate will contribute substantially to his scientific development and further his training in proteomics, RNAi and cancer stem cells biology. The proposed opportunity in the K22 will provide the PI protected time while starting his independent Faculty position and direct him towards a successful career in developing therapeutics for brain tumors. The proposed research will also serve as the foundation for an R01 proposal prior to the end of the second year of this award. Glioma stem cells (GSC) represent a subset population within malignant gliomas, and are highly tumorigenic, intrinsically resistant to current therapies, and capable of self-renewal and differentiation into mature glioma cells (GCs). There are two major subtypes of GSCs: Proneural (PN) and Mesenchymal (MES), the latter being more aggressive and more resistant to conventional treatment. We have identified a subpopulation of GSCs, termed floating cells (FC) that could be isolated from in vitro-cultured glioma cells. These cells possess a stem-like signature with prominent mesenchymal features. They tend to be more aggressive when implanted into the brain of nude mice as compared to their parental GSCs line and have a superior resistance to radiation and therapy. Due to such properties, FC represents an ideal model to identify and test new GBM therapeutics. In Aim 1 of this proposal we will use quantitative mass spectrometry to perform a proteomic analysis on different models of PN and MES GSCs including the FC population. This work will provide a deeper characterization of the FCs and identify differentially expressed proteins/pathways in PN and MES GSCs, which could be exploited to develop new therapeutics. In Aim 2, we will use imaging-based reporters and perform an RNAi screen to identify GSCs modulators which either push GSCs to a more differentiated state, making them susceptible to conventional therapy, or simply kill these cells. Finally we will test a novel gene delivery approach based on extracellular vesicles packaged in adeno-associated virus vectors (vAAV) to deliver our therapeutic RNAi to brain tumors in mice. This work can add valuable information for understanding the biology of this elusive tumor population and plasticity between GSCs subtypes. It also has the potential to identify new vulnerabilities and therapeutic avenues for PN and MES gliomas.